HIV-infected patients fail to fully recover cell-mediated immunity despite HAART. To identify regulatory factors, we studied the phenotype and function of in vitro cytomegalovirus (CMV)-stimulated T cells from HAART recipients. CFSE-measured proliferation showed CD4+ and CD8+ cells dividing in CMV-stimulated cultures. Compared with healthy controls, CMV-stimulated lymphocytes from HAART recipients had lower 3H-thymidine incorporation; lower IFNgamma and TNFalpha production; higher CD4+ CD27- CD28- and CD8+ CD27- CD28- frequencies; lower CD4+ CD25hi; and higher FoxP3 expression in CD8+ CD25hi cells. CMV-specific proliferation correlated with higher IFNgamma, TNFalpha and IL10 levels and higher CD4+ perforin+ and CD8+ perforin+ frequencies. Decreased proliferation correlated with higher CD4+ CD27- CD28- frequencies and TGFbeta1 production, which also correlated with each other. Anti-TGFbeta1 neutralizing antibodies restored CMV-specific proliferation in a dose-dependent fashion. In HIV-infected subjects, decreased proliferation correlated with higher CMV-stimulated CD8+ CD25hi frequencies and their FoxP3 expression. These data indicate that FoxP3- and TGFbeta1-expressing regulatory T cells contribute to decreased immunity in HAART recipients.